Spatial distribution of Mycobacterium tuberculosis mRNA and secreted antigens in acid-fast negative human antemortem and resected tissue

Summary Background The ability to detect evidence of Mycobacterium tuberculosis (Mtb) infection within human tissues is critical to the study of Mtb physiology, tropism, and spatial distribution within TB lesions. The capacity of the widely-used Ziehl-Neelsen (ZN) staining method for identifying Mtb acid-fast bacilli (AFB) in tissue is highly variable, which can limit detection of Mtb bacilli for research and diagnostic purposes. Here, we sought to circumvent these limitations via detection of Mtb mRNA and secreted antigens in human tuberculous tissue. Methods We adapted RNAscope, an RNA in situ hybridisation (RISH) technique, to detect Mtb mRNA in ante- and postmortem human TB tissues and developed a dual ZN/immunohistochemistry staining approach to identify AFB and bacilli producing antigen 85B (Ag85B). Findings We identified Mtb mRNA within intact and disintegrating bacilli as well as extrabacillary mRNA. Mtb mRNA was distributed zonally within necrotic and non-necrotic granulomas. We also found Mtb mRNA within, and adjacent to, necrotic granulomas in ZN-negative lung tissue and in Ag85B-positive bronchiolar epithelium. Intriguingly, we observed accumulation of Mtb mRNA and Ag85B in the cytoplasm of host cells. Notably, many AFB were negative for Ag85B staining. Mtb mRNA was observed in ZN-negative antemortem lymph node biopsies. Interpretation RNAscope and dual ZN/immunohistochemistry staining are well-suited for identifying subsets of intact Mtb and/or bacillary remnants in human tissue. RNAscope can identify Mtb mRNA in ZN-negative tissues from patients with TB and may have diagnostic potential in complex TB cases. Funding Wellcome Leap Delta Tissue Program, Wellcome Strategic Core Award, the 10.13039/100000002National Institutes of Health (NIH, USA), the Mary Heersink Institute for Global Health at UAB, the UAB Heersink School of Medicine.

Table S1.Clinical characteristics of human subjects *A chest X-ray 27 days prior to hospitalization showed no obvious lymphadenopathy with unremarkable heart and lungs.
A chest/abdomen/pelvis CT on hospital day 1 showed no evidence of acute aortic syndrome or central pulmonary thromboembolism.A tree-in bud nodularity in the right middle was noted along with multiple ill-defined upper lobe nodules, suggesting possible infection or inflammation.Metastatic disease was not completely excluded.There was no evidence of retroperitoneal hematoma.Interval increase in simple ascites was noted.Hepatic steatosis was observed with recommendation to correlate with liver enzymes to exclude acute hepatitis.Heterogeneous enhancement spleen was seen, possibly related to arterial phase examination.Small splenic infarcts were not completely excluded.Spleen also appeared enlarged, increased in size from a prior exam, and follow-up was recommended.The patient exhibited right axillary, intrathoracic and extensive intra-abdominal lymphadenopathy.
Due to dyspnea, a chest X-ray on hospital day 5 was performed revealing normal heart and clear lung.Tree-in-bud opacities in the small upper lobe nodules were not identified but may have been too small to see.No acute disease or evidence of pneumonia was noted.
A chest X-ray on the day of death (hospital day 19) showed small layering bilateral pleural effusions, right greater than left and hazy bilateral opacities, right greater than left persist.No pneumothorax was seen with stable cardiomediastinal silhouette.
Culturing of tissue biopsy material was not performed.Antemortem BAL fluid was negative for AFB, but cultures were later found to be positive for Mtb.Urine LAM tests were not performed.Other clinical data include a positive urine histoplasma antigen test on hospital day 14.Seven days prior to hospitalization, a T-spot test gave a borderline result and ACE levels were elevated (152 units/L).
# Patient identity cannot be determined from these anonymized ID numbers

Figure S1 .
Figure S1.RNAscope probe set and tissue control assays.(a) Section of a Hela cell pellet exposed to the positive control RNAscope probe set directed towards human peptidylprolyl isomerase B (PPIB) mRNA showing strong positive signals.(b) Hela cell pellet section exposed to the negative control probe set specific for Bacillus subtilis dihydrodipicolinate reductase (dapB) mRNA is negative for RNAscope signals.(c) Human neonatal lung tissue exposed to the Mtb-specific probe set is negative for RNAscope signals.

Figure S2 .
Figure S2.RNAscope tissue and probe set control assays.(a) Low power image of RNAscope signals from a positive control probe set directed against human peptidylprolyl isomerase B (PPIB) mRNA in a testicular specimen from a patient with TB.Insets; medium power images of boxed areas in (a).(Box 1) Medium power image showing an abundance of RNAscope positive (PPIB) signals.(Box 2) Yellow arrows indicate sparse RNAscope signals in the necrotic area.

Figure S3 .
Figure S3.RNAscope detects Mtb in seminiferous tubules.(a) Low power image of an H&E-stained testicular specimen obtained from a patient with TB.Circled area; seminiferous tubules.Arrows indicate granuloma.(b) Medium power image of a single seminiferous tubule containing numerous Mtb-specific Mtb RNAscope signals.Black arrow indicates the tubule basement membrane.

Figure S4 .
Figure S4.Accumulation of Mtb antigens in extrapulmonary TB tissue.Images of consecutive sections of a seminiferous tubule from a patient with TB.(a) Medium power image of H&E staining.(b) Medium power image showing Mtb-specific RNAscope signals (Note: same image as Figure S3b).(c) Medium power image of Mtb USP-positive IHC staining.(d) Medium power image of ESAT-6-positive IHC staining with a high-power image depicting ESAT-6-positive bacillary shapes (yellow arrow).(e) Medium power image of Ag85B-positive IHC staining with high power image of Ag85B-positive bacillary shapes (yellow arrows).(c, d, e) Note the clear line of demarcation formed by the tubule basement membrane between the accumulated secreted antigens inside the seminiferous tubule and the surrounding tissue confirming the specificity of the antibodies.

Figure S5 .
Figure S5.Mtb Ag85B-positive and -negative bacilli inside seminiferous tubules.(a) and (b) low power images of seminiferous tubules from a patient with TB with combined ZN staining (pink) and Ag85B IHC staining (brown).Insets; medium power images illustrating Ag85B-positive and ZN-positive/Ag85B-negative bacilli.Note: image in (a) is the same image shown in Figure 6a.

Figure S6 .
Figure S6.Ag85B-positive and -negative Mtb bacilli in resected extrapulmonary TB tissue.(A) Low power and (B) medium power images of testicular tissue (outside the seminiferous vesicle) with combined ZN staining (pink) and Ag85B IHC staining (brown).

Figure S7 .
Figure S7.Antemortem biopsy specimens showing evidence of granulomatous inflammation but absence of AFB and fungal organisms.(a-c) Left inguinal lymph node biopsy collected 414 days prior to hospital admission.(d-f) Retroperitoneal lymph node biopsy collected 13 days prior to hospital admission.(a and d) H&E staining.(b and e) ZN staining shows no evidence of AFB.(c and f) Grocott-Gomori Methenamine Silver (GMS) staining shows no evidence of fungus or yeast.Black arrows indicate giant cells associated with granuloma.

Figure S9 .
Figure S9.Autopsy specimens showing acid fast bacilli in multiple tissues.ZN staining of autopsy specimens from (a) Lung, (b) Periaortic abdominal lymph node, (c) Liver, and (d) Bone marrow.Black arrows and area within the oval indicate ZN-positive Mtb bacilli.ZN stain includes methylene blue counter stain.Liver section displayed surface-localized necrotic fibrin exudate that prevented nuclear staining.
Antemortem lymph nodes biopsies revealed granulomatous inflammation on H&E and were negative for AFB on ZN.Antemortem bone marrow revealed focal paratrabecular plasma cells and was not ZN stained clinically due to no granulomas present.Postmortem lung tissue, periaortic abdominal lymph node, liver, and bone marrow all revealed diffuse granulomatous inflammation and were positive for AFB on ZN staining.See clinical imaging notes below*.